BioMicroCenter:Illumina Sequencing
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ILLUMINA MASSIVELY PARALLEL SEQUENCING
The MIT BioMicro Center has four high-throughput Illumina sequencers, including the HiSeq 2000, which are currently being used for a variety of applications, including ChIP-Seq, miRNA sequencing and RNA-seq. Each next-generation sequencer can process up to 7 lanes of samples with multiple samples per lane (if multiplexed), with a data yield of over 25 million reads per lane for the GAIIx and over 120 million reads for the HiSeq (Single read). Read lengths vary, depending on users, and can be sequenced up to 150nt per side on the sequencers.
Illumina sequencing works by binding randomly fragmented DNA to an optical flowcell . Templates are sequenced by incorporating fluorescently labeled nucleotides in a “Sequencing-By-Synthesis” reaction. A detailed description of this process can be found at Illumina's website.
The Genome Analyzers consist of a cluster generation station, a Paired-End module, and a Genome Analyzer, all of which work in concert to generate and analyze flowcells. An overview of the Illumina Genome Analyzer system can be found at the Illumina website.
For an in-depth overview of the Illumina sequencing chemistry, please refer to the following paper:
- Kircher M, Stenzel U, and Kelso J. Improved base calling for the Illumina Genome Analyzer using machine learning strategies. Genome Biol. 2009;10(8):R83. DOI:10.1186/gb-2009-10-8-r83 |
Sample Preparation
For information on Illumina library preparation techniques and services offered by the BMC please visit the Illumina Library Preparation page (please note that this page is currently under development).
Information is also available about multiplexing.
Applications
Illumina currently provides reagents and support for a five major sequencing applications:
Other DNA Sequencing applications: The following applications have been published but do not yet have kits from Illumina.
- Genotyping: Protocols are being developed for detection of SNPs, chromosomal rearrangements and other genotyping applications.
Data Analysis
Each lane of the flowcell should produce between 10 and 25 million DNA fragments as of March 2010. Understanding this data often requires a significant investment in informatics. This is complicated by the fact that many applications require entirely different interpretations of the data. As part of our sequencing service we provide many of the early steps of bioinformatics for different applications. Further data processing can be arranged on a collaborative basis as resources are available. For more information, check out the links below:
- Illumina pipeline - How it works! -- under construction
- Illumina pipeline output formats
- Bioinformatic Consulting
Pricing
Priority for Illumina sequencing is currently available for labs associated with the BioMicro Center Core departments. We are able to do Illumina sequencing for other MIT and non-MIT users as space allows on the sequencers. Full pricing information is available at our price list.
Protocols
Protocols for all of the supported technologies can be found by visiting the Protocols page
QC
Quality control is very important to help optimize the number of reads and quality of data produced. We run the Bioanalyzer and RT-PCR for all submitted cDNA libraries for Illumina sequencing. For information on QC methods and protocols please visit the Sequencing Quality Control page
MIT Core Collaboration
All samples run on the Illumina sequencer are run in batches of 7 flowcell lanes (if many samples are multiplexed into one lane, they count as one for the purpose of completing a batch). In order to optimize our throughput, we have established a collaboration that allows us to move partial flowcells between the various centers at MIT. For users with less then 4 samples, their samples may be moved between the BioMicro Center, the Whitehead Institute Center for Genome Technologies and the Koch Institute Biopolymer Center. Samples will be moved only to fill out runs or to expedite processing. The Centers are committed to working together to maintain consistent quality between the different cores so you should see no difference whether your samples are run in BioMicro or at one of our sister centers. Transfers are only available for members of the MIT community.
View current samples queuing for Illumina.
All questions about Illumina Sequencing can be directed to Kevin Thai at kthai@mit.edu.
Initial page written by Summeet Gupta at the WI-CGT
